Figure 5.

ROCK-Driven Myosin II Activity in Immunotherapy-Resistant Melanoma

(A) Top, schematic of cell lines. Bottom, p-MLC2 immunoblots after treatment: n = 7 (G); n = 3 (H).

(B) Survival of patient no. 26 cells treated for 10 days (n = 4).

(C) Top, schematic of experiment. Bottom, survival of 4434 and 5555 anti-PD-1/non-responder (NR) lines versus responder (Resp) after a 3-day treatment (n = 3, 5555; n = 4, 4434).

(D) p-MLC2 and p-ERK1/2 immunoblots of patient no. 58 (n = 4) and no. 33 (n = 3) cells after treatment.

(E) Images and quantification of cell survival on collagen I for 7 days (n = 3).

(F) p-MLC2 and p-ERK1/2 immunoblots of patient no. 62T3 cells after treatment (n = 3).

(G) Survival of patient no. 62T3 cells after a 10-day treatment (n = 3).

(H) p-MLC2 and p-ERK1/2 immunoblots of patient no. 2 cells after treatment (n = 5).

(I) Cell morphology of patient no. 2 cells on collagen I after treatment. n = 70 cells (dots) from 2 experiments. Scale bar, 50 μm.

(J) Survival of patient no. 2 cells as spheroid-forming ability on collagen I for 16 days (n = 3); Scale bar, 100 μm.

(K) Survival after a 10-day blebbistatin treatment (n = 3).

(L) Survival 8 days after gene depletion by RNAi (n = 3; n = 4 patient no. 2 MYL12B-ROCK1/2, no. 62T3 MYL9; n = 5 no. 62T3 MYL12B). Average percentage mRNA KD (percentage decrease versus control) by qRT-PCR is shown. Vertical line in (D, F, and H): cell line establishment.

(A–J) ROCKi GSK269962A, H1152; (F–K) BRAFi PLX4720.

(A, D, F, H, and I) 24 h treatment.

Graphs show mean ± SEM and individual data points (circle) except boxplot in I (median, center line; interquartile range, box; min-max, whiskers). p values by one-way ANOVA with Tukey's (B, C, G, and J) or Dunnett's correction (L, myosin genes); Kruskal-Wallis with Dunn's correction (I), t test with Welch's correction (E, K, and L, ROCK); ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; n.s., not significant. See also Figure S5.